Auxin-Induced Degradation of DPY30 and RBBP5

For the generation of the auxin-inducible degradation system for DPY30 sgRNA targeting the stop-codon region were cloned into eSpCas9(1.1)-T2A-eGFP. Left and right homology arms, as well as the mAID-T2A-BFP middle part were ligated into a modified pUC19 vector backbone (a gift from S. Pollard) using the In-Fusion cloning kit (Takara, 638910). mES cells were co-transfected with sgRNA- and donor-vector using Lipofectamine 3000 and sorted 48 h later for GFP/BFP-double-positive cells. Homozygous clones were then transfected with pPB-hygro-OsTIR1-P2A-mCherry and pBase plasmids and selected with 100 µg ml⁻¹ hygromycin B (Thermo Fisher Scientific, 10687010). For the generation of the endogenous dTAG-inducible degradation system for RBBP5, sgRNA targeting the stop codon region were co-transfected into the cells and contained the following elements: left and right homology arms, as well as FKBP12(F36V), 2× HA tags, P2A and a neomycin-resistance gene. The transfected cells were selected with 100 µg ml⁻¹ Geneticin selective antibiotic (G418 Sulfate) (Thermo Fisher Scientific, 10131027), single-cell sorted to obtain clonal cell lines and screened for correct biallelic integration. All homozygous insertions and knock-ins were confirmed by Sanger sequencing and western blotting. A list of the oligos and the sequences of the sgRNAs is provided in Supplementary Table 1.